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Synthesis of Nucleoside 5′-Tetraphosphates Containing Terminal Fluorescent Labels via Activated Cyclic Trimetaphosphate Samy Mohamady and Scott D. Taylor* Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada S Supporting Information *
ABSTRACT: 2′-Deoxynucleotide 5′-tetraphosphates in which a fluorescent label is attached to the terminal phosphate are used as key reagents in high-throughput DNA sequencing techniques and in single nucleotide polymorphism typing assays. We demonstrate that this class of compounds can be prepared by reacting fluorophores such as 7-hydroxy-4-methylcoumarin, methylfluorescein, fluorescein and resorufin with an activated form of cyclic trimetaphosphate to give intermediate 11. Reaction of 11 with 2′-deoxynucleoside 5′-monophosphates or a nucleoside 5′-monophosphate gave the target compounds in good yield.
T
for preparing dinucleoside tetraphosphates (Np4N’s). In one approach (route 1), a 2′-nucleoside 5′-triphosphate (5′-dNTP) is reacted with carbonyldiimidazole (CDI) followed by reacting the resulting imidizolide with a phosphorylated fluorescent dye.8 Alternatively, a phosphorylated dye is activated with CDI followed by reaction with a 5′-dNTP (route 2).4 In another approach (route 3), a 5′-dNTP is reacted with DCC to give a cyclic trimetaphosphate nucleotide derivative, which is ring opened with orthophosphate to give a nucleoside 5′tetraphosphates (Np4).5 This species is activated and then reacted with a dye. All of these approaches require expensive 5′dNTP’s as substrates and, in the case of routes 1 and 2, the synthesis or purchase of expensive phosphorylated dyes.9 Besides the routes described in Scheme 1, other approaches have been developed for preparing conjugates of dinucleoside tetraphosphates, which could potentially be used for preparing δ-labeled nucleoside 5′-tetraphosphates. Methods employing 5′-chloroquinolyl esters,10 5′-cyclosaligenyl phosphates,11 and 5′-salicylphosphites have been reported.12 In each case the prior preparation of an activating agent and/or the protection of the secondary hydroxyl or amino groups on the nucleoside and/or isolation of the activated nucleoside is required. A solid phase method has recently been described; however, this method requires the multistep synthesis of a polymer with a unique linker and multistep syntheses of polyphosphites prior to the solid phase chemistry.13 Enzymatic methods have also been used for the synthesis of Np4N’s; however, this approach is limited by the substrate specificity of the enzymes.14 Ludwig and Eckstein developed a method for preparing nucleoside
erminal phosphate-labeled nucleotides have been used as tools and probes in biochemistry and biotechnology for many years.1−3 Very recently there has been considerable interest in 2′-deoxynucleoside 5′-polyphosphates in which a fluorescent label is attached to the terminal phosphate. Such nucleotides are used as key reagents in high-throughput DNA sequencing techniques and in single nucleotide polymorphism typing assays.4−8 Central to the success of these methodologies is the ability of the terminally labeled nucleotides to as act as substrates for DNA polymerase. Nucleotides bearing more than three linear 5′-phosphates, such as δ-labeled nucleoside 5′tetraphosphates (general structure 1 in Scheme 1), are used, as it has been shown that such nucleotides are much better substrates for DNA polymerases than the corresponding γlabeled 5′-triphosphates.4 The synthesis of δ-labeled nucleoside 5′-tetraphosphates has been achieved by several routes (Scheme 1). These approaches are based upon routes that were developed and are widely used Scheme 1. Literature Routes to Nucleoside 5′Tetraphosphates Bearing Terminal Fluorescent Dyes
Received: January 9, 2014 Published: February 5, 2014 © 2014 American Chemical Society
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−15.2 ppm (d) and two apparent triplets at approximately −20.0 to −20.3 ppm) along with other unidentified impurities. Optimal conditions were developed, which consisted of 1.3 equiv of HMC, 4 equiv of DABCO and 1.1 equiv of MgCl2. The HMC was consumed in about 5 h. After quenching with triethylammonium acetate and purification by reversed-phase column chromatography, compound 6 was obtained in a 45% yield. The modest yield obtained with the approach outlined in Scheme 3 prompted us to examine the reverse procedure in which the coumarin is reacted with activated TriMP followed by the addition of NMP (Scheme 4). To ensure complete
triphosphates using salicylchlorophosphite as an activating agent.15 To the best of our knowledge this methodology has never been used for preparing conjugates of nucleoside tetraphosphates. Although it could potentially be used for this purpose it would require the protection of the nucleoside and the use of an oxidant, which could affect the fluorophore. We recently described a novel route to the synthesis of Np4’s and dinucleoside 5′-pentaphosphates (Np5N’s), which involves reacting a nucleoside 5′-monophosphate with activated trimetaphosphate (TriMP, 3) to give intermediate 4 (Scheme 2).16 Hydrolysis of this intermediate yielded the Np4’s, while Scheme 2. Synthesis of Np4’s and Np5N’s via Activated TriMP (3)
Scheme 4. Synthesis of δ-Labeled Nucleoside 5′Tetraphosphates via Intermediate 11
reaction with a nucleoside 5′-monophosphate (5′-NMP) gave Np5N’s in high yield and purity. The success of these studies prompted us to examine whether intermediates of type 4 and their 2′-deoxy analogues could be reacted with other nucleophiles, such as a fluorescent dye, to give a rapid and straightforward route to δ-labeled nucleoside 5′-tetraphosphates. 7-Hydroxy-4-methylcoumarin (HMC, Scheme 3) was chosen as a model fluorescent dye, as it is relatively inexpensive and
phosphorylation of the OH group of HMC and to prevent formation of the dicoumarin triphosphate, we employed a 1.8fold excess of activated TriMP. Hence, 1.8 equiv of mesitylenesulfonyl chloride was added to a solution of 2.0 equiv of TriMP (2) and 6 equiv of DABCO, and the mixture was stirred for 1 min, and then 1.0 equiv of HMC was added. The reaction turns from a clear solution to a white turbid mixture after about 45 min. All of the coumarin was consumed in about 2 h as determined by TLC. No reaction occurred in the absence of DABCO, and it was found that NMI was not necessary for reaction to occur. This mixture was then added dropwise to a cooled (ice bath) solution of 1.6 equiv of MgCl2 and 2.5 equiv of 5′-AMP as its tetrabutylammonium salt in DMF. The ice bath was removed, and the progress of the reaction was monitored by withdrawing samples, quenching them with a 5% solution of EDTA in triethylammonium acetate buffer (pH 7.0), and then analyzing them by 31P NMR. Peaks corresponding to 6 were clearly evident. After 3 h, peaks corresponding to 6 no longer increased in intensity, and 6 was the dominant product. The reaction was quenched with triethylammonium acetate buffer (pH 7.0), and the resulting solution was washed with chloroform, and magnesium ions were removed by Chelex resin. The 31P NMR of the crude material exhibited peaks corresponding to product as well as peaks corresponding to TriMP, unreacted 5′-AMP, and minor peaks that we attributed to Ap5A15 (for example, see Figure S1 in the Supporting Information). With the activated TriMP and 5′-AMP being in considerable excess to HMC, it was expected for a considerable amount of Ap5A byproduct to have been
Scheme 3. Synthesis of Compound 6 via Intermediate 5
readily available. We initially attempted to prepare the adenosine derivative 6 by reacting intermediate 5, prepared using our previously reported procedure,16 with HMC in the presence of MgCl2. This did not result in consumption of the HMC as determined by TLC. However, when an excess of sterically hindered organic base such as DABCO was present the coumarin was consumed and the formation of 6 was evident by 31P NMR (characteristic peaks: −9.3 ppm (d), 2309
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DABCO (approximately 1:1.3:1.3:4) in CH3CN after 2 h showed mainly a doublet at −20.7 ppm and a triplet at −24 ppm (Figure S2 in the Supporting Information). The 31P NMR spectrum of this mixture after having been quenched with water showed doublets at −4.0 and −14.5 ppm and a triplet at −20 ppm (see Figure S3 in the Supporting Information), and these peaks can be attributed to compound 15 formed by hydrolysis of intermediate 11 (Scheme 6).17
produced and little or no unreacted 5′-AMP or TriMP to be present. Since this was not the case, we suggest that the reaction of the 5′-AMP with any activated TriMP intermediates that might be formed during the reaction (vide infra) is slower than the reaction of 5′-AMP with intermediate 11 in the absence of NMI and/or in the presence of DABCO. Compound 6 was obtained in an 80% yield after purification by reversed-phase chromatography using tributylammonium acetate buffer in methanol/water and conversion to its tetraammonium salt using Dowex 50W resin (NH4+ form). It is worthy of note that the Ap5A byproduct exhibited a very different retention time from 6 and so was easily removed. This procedure was then applied to the four common deoxynucleotides, which gave the labeled products 7−10 in yields of 80− 82%. Purification of these compounds was equally straightforward. To ascertain if this methodology can be applied to the synthesis of nucleoside tetraphosphate conjugates bearing other fluorophores, we prepared adenosine tetraphosphate conjugates in which the terminal phosphate was labeled with resorufin, methyl fluorescein and fluorescein (compounds 12−14, Scheme 5). These compounds were prepared in good yield
Scheme 6. Formation of Triphosphate 15 by Hydrolysis of Intermediate 11
Although we believe that the reaction proceeds via intermediate 11, the route by which this intermediate is formed is not clear. The 31P NMR spectrum of a mixture of TriMP, DABCO, and mesitylenesulfonyl chloride (1:3:0.9) in acetonitrile after 5 min shows a doublet at −6.3 ppm and a triplet at −20.6 ppm (see Figure S4 in the Supporting Information). However, after about one hour these peaks are no longer present. The mixture becomes turbid, and the 31P NMR spectrum shows a triplet at −31 ppm and a multiplet at around −20 ppm, possibly consisting of overlapping doublets and triplets (see Figure S5 in the Supporting Information). Since it takes 2 h for the coumarin to be completely consumed, these data suggest that intermediate 11 might be formed by HMC reacting with more than one intermediate. It is possible that the initially formed mixed anhydride 1616 reacts rapidly with DABCO to give intermediate 17, and this species may react with HMC to give intermediate 11 (Scheme 7).
Scheme 5. Synthesis of Adenosine-5′-tetraphosphate δLabeled with Resorufin, Methyl Fluorescein, and Fluorescein
Scheme 7. Possible Pathway for Formation of Intermediate 11
with compounds 13 and 14 obtained as mixtures of diastereomers and/or as a mixture of the lactone and free acid forms. The reactions of the trimetaphosphorylated derivatives of these dyes with 5′-AMP took longer (overnight as opposed to 3 h in case of HMC). For the methylfluorescein and fluorescein derivatives 13 and 14, the yield varied significantly with the amount of DABCO present. When using 6 equiv of DABCO, compound 14 was obtained in a 54% yield together with a significant quantity of disubstituted product ((O3P)3O-fluorescein-O(PO3)3). Adding just one more equivalent of DABCO (7 equiv) resulted in a yield of 68%, and the amount of disubstituted product was significantly reduced. On the other hand, using 6 equiv of DABCO gave a low yield of compound 13 (42%). Increasing the DABCO to 7 equiv reduced the yield to 25%, while using 4 equiv of DABCO gave a 74% yield of compound 13. The effect of DABCO on the yields of 13 and 14 may be a result of DABCO affecting the relative amounts of lactone and free acid forms of fluorescein and methyl fluorescein substrates. 31 P NMR data suggests that intermediate 11 is indeed formed during the reaction. The 31P NMR spectrum of a mixture of HMC, TriMP, mesitylenesulfonyl chloride, and
Moreover, dimeric or higher order polyphosphates might be formed, and these species may also be capable of reacting with HMC to somehow give intermediate 11. Further studies will be required to determine this. In summary, a novel and efficient approach to the synthesis of δ-labeled nucleoside 5′-tetraphosphates in which the terminal phosphate is labeled with a fluorescent dye was developed. The procedure is more rapid than previously reported methods,4,5,8 it utilizes relatively inexpensive or easily 2310
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NMR spectrum of the residue indicated that there were 1.3 tributylammonium ions per nucleoside monophosphate. The nucleotide was subjected to a high vacuum for 3 h. The flask was removed under Ar, the nucleotide was dissolved in dry DMF, and 4 Å molecular sieves were added. This solution was allowed to stand for at least 3 h hour prior to the coupling reaction. δ-(4-Methyl-7-coumarinyl) adenosine 5′-tetraphosphate tetraammonium salt (6). Method A (Scheme 3). To a mixture of 2 (0.405 mmol, 390 mg) in acetonitrile (4.5 mL) and Nmethylimidazole (0.12 mmol, 97 μL) was added mesitylenesulfonyl chloride (MS-Cl, 0.36 mmol, 80 mg) at room temperature, and the reaction mixture was stirred for 15 min. This solution was withdrawn via syringe and injected dropwise over 1 min into a cooled flask (ice bath) containing tetrabutylammonium salt of AMP (0.27 mmol, 206 mg) in DMF (3 mL). 7-Hydroxy-4-methylcoumarin (0.351 mmol, 62.0 mg) and DABCO (0.81 mmol, 91 mg) were added to the reaction flask followed by anhydrous magnesium chloride (0.315 mmol, 30 mg). The ice bath was then removed, and the reaction mixture was stirred at room temperature for 5 h. The reaction mixture was then cooled in ice and quenched with 100 mM triethylammonium acetate buffer (pH 7.0, 6 mL) and then washed with chloroform (3 × 10 mL). Chelex resin (ca. 0.1 g) was added, and the mixture stirred for 1 min and then filtered through a cotton plug. The filtrate was purified by reversed-phase column chromatography using a gradient of 75% buffer A (10 mM tributylamine-30 mM acetic acid), 25% buffer B (methanol, 15 mM tributylamine) to 40% buffer A, 60% buffer B over 55 min. The flow rate was 25 mL/min and monitored at 265 and 280 nm (tR = 40 min). Fractions containing the desired product were pooled and concentrated by high vacuum rotary evaporation, and the residue was dissolved in water and repeatedly freeze-dried until the 1H NMR spectrum indicated that no residual buffer was present (four times). The resulting white powder was converted to its ammonium salt using a Dowex-50-W ion-exchange resin in NH4+ form to afford after lyophilization, 99 mg (45%) of the tetraammonium salt of compound 6: 1H NMR (D2O, 300 MHz) δ 2.10 (s, 3H, CH), 4.14 (s, 2H), 4.19 (s, 1H),4.35 (s, 1H),4.46 (t, J = 4.5 Hz, 1H), 5.75 (d, J = 4.3 Hz, 1H), 5.89 (s, 1H), 6.8 (s, 1H), 6.92 (d, J = 8.6 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 7.88 (s, 1H), 8.15 (s, 1H); 13C NMR (D2O, 75 MHz) δ 17.8, 65.0 (d, J = 5.2 Hz), 70.1, 74.5, 83.5 (d, J = 9.2 Hz), 86.9, 107. 9, 108.0, 111.5, 115.6, 117.4 (d, J = 4.5 Hz), 117.6, 125.9, 140.0, 147.8, 149.8, 152.8, 154.3 (d, J = 6.6 Hz), 155.5, 163.7; 31P NMR (D2O, 121 MHz) δ −9.02 (d, J = 16.5 Hz), −14.52 (d, J = 14.4 Hz), −21.02 (m); HRMS (ESI−) m/z = 743.99319, C20H22O18N5P4 [M − H]−, requires 743.99158. General Method for the Preparation of δ-(4-Methyl-7coumarinyl) nucleoside 5′-tetraphosphate tetraammonium salts (6−10), δ-(3H-Phenoxazin-3-one-7-yl) adenosine 5′tetraphosphate tetraammonium salt (12), δ-(6′-Methoxy-3Hspiro[2-benzofuran-1,9′-xanthen]-3′-yl) adenosine 5′-tetraphosphate tetraammonium salt (13), and δ-(6′-Hydroxy-3Hspiro[2-benzofuran-1,9′-xanthen]-3′-yl) adenosine 5′-tetraphosphate tetraammonium salt (14). Method B (Schemes 4 and 5). To a mixture of 2 (0.54 mmol, 520 mg) and DABCO (1.62 mmol, 180 mg for compounds 6−12, 1.08 mmol, 121 mg for compound 13, 1.89 mmol, 212 mg for compound 14) in dry acetonitrile (6 mL) was added MS-Cl (0.48 mmol, 106 mg for compounds 6−10, 12, and 0.54 mmol, 118 mg for compounds 13 and 14). The mixture was stirred at room temperature for 1 min, and then 7-hydroxy-4-methylcoumarin (HMC), resorufin, 3′-O-methylfluorescein, or fluorescein (0.27 mmol, 48, 58, 93, and 90 mg, respectively) were added, and the reaction mixture was allowed to stir at room temperature for 2 h (6 h in case of 13). For compounds 6−10, a white turbidity appeared after about 45 min. For compound 12, the turbid solution was orange-red, and for compounds 13 and 14 it was yellowish-brown. The reaction mixture was then withdrawn by a syringe and injected dropwise over 1 min into a cooled (ice bath) solution of NMP or 2′-dNMP (0.675 mmol) and anhydrous magnesium chloride (0.42 mmol, 40 mg) in DMF (7.5 mL). The ice bath was removed, and the reaction mixture was allowed to stir at room temperature for 3 or 15 h in cases of 12−14. The turbidity disappears except when using 2′-dCMP or 2′-dGMP. The reaction
prepared NMP’s as opposed to more expensive NTP’s, and the target compounds are obtained in excellent yields. These studies also demonstrate that activated TriMP is capable of reacting with nucleophiles other than phosphates or excess water and so should prove to be a route to other types of δlabeled nucleoside tetraphosphates and could also be a direct route to nucleoside 5′-triphosphates using nucleosides as nucleophiles.
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EXPERIMENTAL SECTION
General Information. All reagents and starting nucleotides were obtained from commercial sources unless stated otherwise. Strictly anhydrous conditions are required to obtain the reported yields. Acetonitrile and DMF were distilled from calcium hydride. NMethylimidazole (NMI) was distilled from sodium hydroxide and stored over 4 Å molecular sieves. Methylfluorescein was prepared according to literature procedures and crystallized from boiling ethanol.18,19 All reactions were conducted under an inert atmosphere of Ar in oven-dried glassware. All NMR spectra were recorded using D2O as solvent. For 1H NMR spectra, chemical shifts are reported in ppm relative to the solvent residual peak (δ 4.79). For protondecoupled 13C NMR spectra, chemical shifts are reported in ppm relative to CH3OH in D2O (δ 49.5, external standard). For protondecoupled 31P NMR, chemical shifts are reported in ppm relative to aqueous 85% H3PO4 (δ 0 ppm, external standard). The tri(tetrabutylammonium) salt of trimetaphosphate (2) was prepared as previously described.16 Preparative reversed phase chromatography was performed using a Biotage Isolera One Flash purification system equipped with a C-18 reverse phase preparative Biotage 30 g column. Negative ion high resolution electrospray mass spectra were obtained using a high resolution, accurate mass Orbitrap mass spectrometer. Preparation of the Tetrabutylammonium Salts of 5′-AMP, 5′-dAMP, 5′-dTMP, and 5′-dCMP. The free acids or free acid monohydrates of 5′-AMP, 5′-dAMP, 5′-dTMP, and 5′-dCMP (0.5 g) were dissolved in distilled deionized water (50 mL) and titrated to pH 7.0 with a dilute solution of tetrabutylammonium hydroxide.20 The solutions were concentrated by high vacuum rotary evaporation to approximately one-seventh the original volume and lyophilized. The lyophilized powders were stored in the freezer. An equal amount of dry acetonitrile and dry toluene was added to round-bottom flasks containing 0.675 mmol (519 mg of AMP, 508 mg of 2′-dAMP, 502 mg of 5′-dTMP, and 492 mg of 5′-dCMP) of these nucleotides, and the solutions were concentrated by rotary evaporation to dryness. This was repeated two more times. The 1H NMR spectra of these nucleotides indicated that there was 1.7 tetrabutylammonium ions per nucleoside monophosphate. The nucleotides were subjected to a high vacuum for 3 h. The flasks were removed under Ar, the nucleotides were dissolved in dry DMF, and 4 Å molecular sieves were added. These solutions were allowed to stand for at least 3 h hour prior to the coupling reactions. Preparation of the Tributylammonium Salt of 2′-dGMP. Using the above procedure to prepare the tetrabutylammonium salt of 2′-dGMP from commercially available 2′-dGMP disodium salt dihydrate resulted in its decomposition upon conversion to its free acid and titration with tetrabutylammonium hydroxide. Hence the trin-butylammonium salt was prepared using a procedure similar to that developed by Gibson and Leonard.21 An aqueous solution of 2′-dGMP disodium salt dihydrate (0.5 g in 20 mL of distilled deionized water) was applied to a Dowex-50-W column (8−10 g) in its pyridinium form. The eluate was collected in a flask containing 20 mL of ethanol and 1.0 mL of tributylamine. The column was washed with water (3 × 30 mL) while collecting the eluate in the above-mentioned flask. The resulting solution was stirred for 10 min and then concentrated by high vacuum rotary evaporation to about one-seventh of the original volume and then lyophilized. The lyophilized powder was stored in the freezer. An equal amount of dry acetonitrile and dry toluene was added to a round-bottom flask containing 0.675 mmol (396 mg) of the tributylammonium salt, and the solution was concentrated by rotary evaporation to dryness. This was repeated two more times. The 1H 2311
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Note
43 min; 1H NMR (D2O, 300 MHz) δ 4.14 (br, 3H), 4.37 (brs, 1H), 4.42 (t, J = 5.0 Hz, 1 H), 5.75 (J = 5.0 Hz, 1 H), 6.16 (s, 1H), 6.96 (d, J = 9.7 Hz, 1 H 1H), 7.15 (s, 1H), 7.24 (t, J = 10.0 Hz, 2H), 7.52 (d, J = 8.9 Hz, 1H), 7.88 (s, 1H), 8.08 (s, 1H); 13C NMR (D2O, 75 MHz) δ 65.1 (d, J = 5.4 Hz), 70.2, 74.8, 83.5 (d, J = 8.7 Hz), 86.8, 105.6, 107.8 (d, J = 5.4 Hz), 117.9, 119.2 (d, J = 4.3 Hz), 129.2 130.7, 133.6, 134.4, 139.4, 144.6, 145.2, 148.2, 150.3, 151.8 154.3, 156.2 (d, J = 7.6 Hz), 188.2; 31P NMR (D2O, 121 MHz) δ −8.77 (d, J = 10.9 Hz), −14.72 (d, J = 14.2 Hz), −20.61 (s); HRMS (ESI−) m/z = 780.98726, C22H21O18N6P4 [M − H]−, requires 780.98683. δ-(6′-Methoxy-3H-spiro[2-benzofuran-1,9′-xanthen]-3′-yl) adenosine 5′-tetraphosphate tetraammonium salts (13). Obtained as a mixture of diastereomers in 74% yield (196 mg): tR = 38 min; 1H NMR (D2O, 300 MHz) δ 3.53 (s, 1H), 4.07 (br, 3H), 4.35 (s, 2H), 5.73 (dd, J = 3.7, 3.6 Hz, 1H), 6.10 (brs, 2H), 6.46 (s, 1H), 6.57 (brm, 1H), 6.67 (brs, 1H), 6.83 (d, J = 8.1 Hz, 1H), 7.07 (s, 1H), 7.39 (brt, J = 8.4 Hz, 3H), 7.79 (d, J = 5.7 Hz, 1H), 7.90 (s, 1H), 8.26 (s, 1H); 13C NMR (D2O, 75 MHz) δ. 55.5, 65.0, 70.0 (d, J = 4.2 Hz), 74.9, 83.5 (overlapping doublets), 84.1 (d, J = 4.6 Hz), 87.4, 100.69, 100.75, 108.8, 109.8, 111.2, 113.8, 116.6, 117.9, 123.5, 124.8, 124.9, 125.0, 128.3, 128.8, 128.9, 130.2, 135.9, 140.5, 147.9, 148.3, 151.3, 151.5, 152.1, 152.2, 152.3, 153.5 (d, J = 6.5 Hz), 160.7, 171.56, 171.63; 31 P NMR (D2O, 121 MHz) δ −9.05 (d, J = 15.2 Hz), −14.67 (d, J = 15.5 Hz), −20.95 (s); HRMS (ESI−) m/z = 914.02907, C31H28O20N5P4 [M − H]−, requires 914.02836. δ-(6′-Hydroxy-3H-spiro[2-benzofuran-1,9′-xanthen]-3′-yl) adenosine 5′-tetraphosphate tetraammonium salts (14). Obtained as mixture of isomers (lactone and free acid forms) in 68% yield (178 mg): tR = 47 min; 1H NMR (D2O, 300 MHz) δ 4.09 (s, 3H), 4.29 (s, 2H), 5.76 (brd, 1H), 6.28 (m, 3H), 6.56 (m, 1H), 6.76 (brs, 2H), 6.99 (d, J = 9.0 Hz, 1H), 749 (brs, 2H), 7.82 (brs, 1H), 7.95 (s, 1H), 8.24 (d, J = 5.9 Hz, 1H); 13C NMR (D2O, 75 MHz) δ 64.8, 69.8, 74.9, 83.3 (overlapping doublets), 87.6, 102.4, 108.4, 109.6, 113.1, 113.2, 113.8, 113.9, 116.7, 118.0, 124.0, 125.0, 125.06, 125.7, 125.8, 128.9, 129.9, 130.1, 135.6, 140.8, 147.3, 147.6, 151.0, 151.2, 151.3. 151.85, 151.94, 153.48, 153.53, 159.4, 159.6, 171.9, 172.0; 31P NMR (D2O, 121 MHz) δ −8.80 (d, J = 14.3 Hz), −14.08 (d, J = 12.8 Hz), −20.66 (s); HRMS (ESI−) m/z = 900.01384, C30H26O20N5P4 [M − H]−, requires 900.01271.
mixture was then cooled in an ice bath and quenched by adding triethylammonium acetate buffer (pH 7.0, 8 mL). The solution was washed with chloroform (3 × 10 mL). Chelex resin (ca. 0.1 g) was added, and the mixture was stirred for 1 min and then filtered through a cotton plug. The filtrate was purified by reversed-phase chromatography using a linear gradient of 75% buffer A (10 mM tributylamine-30 mM acetic acid), 25% buffer B (15 mM tributylamine in MeOH) to 40% buffer A, 60% buffer B over 55 min, except for compound 13, where a linear gradient of 60% buffer A, 40% buffer B to 30% buffer A, 70% buffer B over 55 min was used. The flow rate was 25 mL/min, and eluate was monitored at 265 and 280 nm. Fractions containing the desired product were pooled and concentrated by high vacuum rotary evaporation, and the residue was dissolved in water and repeatedly freeze-dried until the 1H NMR spectrum indicated that no residual buffer was present (four times). The resulting white powder (except for compound 12, which was a reddish brown powder) was converted to its ammonium salt using a Dowex-50-W ion-exchange resin in NH4+ form. δ-(4-Methyl-7-coumarinyl) adenosine 5′-tetraphosphate, tetraammonium salt (6). Obtained in 80% yield (176 mg). Characterization data were identical to that reported above using method A. δ-(4-Methyl-7-coumarinyl) 2′-deoxyadenosine 5′-tetraphosphate, tetraammonium salt (7). Obtained in 80% yield (172 mg): tR = 32 min; 1H NMR (D2O, 300 MHz) δ 2.05 (s, 3H), 2.31−2.51 (m, 2H), 4.01−4.08 (m, 3H), 4.56 (s, 1H), 5.83 (s, 1H), 6.05 (t, J = 6.8 Hz, 1H), 6.82 (s, 1H), 6.94 (dd, J = 9.0, 1.8 Hz, 1H), 7.25 (d, J = 9.0 Hz, 1H),7.79 (s, 1H), 8.07 (s, 1H); 13C NMR (D2O, 75 MHz) δ 17.8, 39.2, 65.3 (d, J = 5.4 Hz), 71.0, 83.5, 85.4 (d, J = 8.9 Hz), 107. 8, 107.9, 111.4, 115.5, 117.4 (d, J = 4.5 Hz), 125.9, 139.6, 147.4, 150.5, 152.6, 153.4, 154.2 (d, J = 6.8 Hz), 155.3, 163.5; 31P NMR (D2O, 121 MHz) δ −9.06 (d, J = 15.7 Hz), −14.77 (d, J = 12.8 Hz), −21.02 (s); HRMS (ESI−) m/z = 727.99745, C20H22O17N5P4 [M − H]−, requires 727.99666. δ-(4-Methyl-7-coumarinyl) 2′-deoxy-cytosine 5′-tetraphosphate, tetraammonium salt (8). Obtained in 81% yield (169 mg): tR = 31 min; 1H NMR (D2O, 300 MHz) δ 2.01 (m, 1H), 2.18 (m, 1H), 2.24 (s, 3H), 3.98 (br, 3H), 4.38 (s, 1H), 5.88 (br, 2H), 6.06 (s, 1H), 7.06 (br, 2H), 7.52 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 6.0 Hz, 1H); 13 C NMR (D2O, 75 MHz) δ 17.9, 39.4, 65.1 (d, J = 5.2 Hz), 70.5, 85.6 (d, J = 9.3 Hz), 86.0, 95.4, 108.2 (d, J = 5.1 Hz), 111.8, 116.1, 117.6 (d, J = 5.0 Hz), 126.4, 142.8, 150.6, 153.2, 154.6 (d, J = 6.8 Hz), 155.8, 160.6, 164.0; 31P NMR (D2O, 121 MHz) δ −8.96 (d, J = 9.8 Hz), −14.77 (d, J = 10.6 Hz), −20.94 (s); HRMS (ESI−) m/z = 703.98597, C19H22O18N3P4 [M − H]−, requires 703.98543. δ-(4-Methyl-7-coumarinyl) 2′-deoxythymidine 5′-tetraphosphate, tetraammonium salt (9). Obtained in 82% yield (174 mg): tR = 34 min; 1H NMR (D2O, 300 MHz) δ 1.64 (s, 3H), 2.07−2.13 (m, 2H), 2.27 (s, 3H), 3.96 (s, 1H), 4.04 (brs, 2H), 4.43 (brs, 1H), 6.01 (t, 1H), 6.09 (s, 1H), 7.12 (br, 2H), 7.38 (s, 1H), 7.55 (d, J = 6.0 Hz, 1H); 13C NMR (D2O, 75 MHz) δ 11.5, 17.8, 39.5, 65.3 (d, J = 5.5 Hz), 70.7, 84.6 85.0 (d, J = 9.2 Hz), 108.1 (d, J = 5.2 Hz), 111.2, 111.7, 116.0, 117.6 (d, J = 5.0 Hz), 126.3, 136.8, 151.1, 153.2, 154.6 (d, J = 6.9 Hz), 155.8, 163.9, 165.8; 31P NMR (D2O, 121 MHz) δ −9.26 (d, J = 15.9 Hz), −14.78 (d, J = 16.3 Hz), −21.09 (s); HRMS (ESI−) m/z = 718.98522, C20H23O19N2P4 [M − H]−, requires 718.98510. δ-(4-Methyl-7-coumarinyl) 2′-deoxy-guanosine 5′-tetraphosphate tetraammonium salt (10). Obtained in 82% yield (180 mg): tR = 36 min; 1H NMR (D2O, 300 MHz) δ 2.10 (s, 3H), 2.34 (m, 1H), 2.42 (m, 1H), 4.06 (m, 3H), 4.54 (s, 1H), 5.90 (brs, 2H), 6.90 (s, 1H), 7.00 (d, J = 8.6 Hz, 1H), 7.33 (d, J = 8.6 Hz, 1H), 7.99 (s, 1H); 13C NMR (D2O, 75 MHz) δ 17.7, 39.0, 65.2 (d, J = 5.4 Hz), 70.8, 83.9, 85.4 (d, J = 9.0 Hz), 107. Eight (d, J = 5.5 Hz), 111.4, 113.0, 117.3 (d, J = 4.5 Hz), 126.0, 136.3, 149.7, 152.8, 153.5, 154.3 (d, J = 6.8 Hz), 155.4, 156.6, 163.5; 31P NMR (D2O, 121 MHz) δ −9.05 (d, J = 15.3 Hz), −14.77 (d, J = 13.9 Hz), −21.03 (s); HRMS (ESI−) m/z = 743.99260, C20H22O18N5P4 [M − H]−, requires 743.99158. δ-(3H-Phenoxazin-3-one-7-yl) adenosine 5′-tetraphosphate tetraammonium salts (12). Obtained in 80% yield (184 mg): tR =
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ASSOCIATED CONTENT
S Supporting Information *
Figures S1−S5. Copies of 1H, 13C, and 31P NMR spectra for compounds 6−10 and 12−14. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS S.D.T. is grateful to the Natural Sciences and Engineering Research Council (NSERC) of Canada for support of this work through a Discovery Grant.
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REFERENCES
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